Visco-supplement compositions, and methods of use thereof

ABSTRACT

Visco-supplement compositions derived from a transcellular fluid, such as human amniotic fluid, human aqueous humor fluid, or human vitreous fluid are described. Also described are methods for treating inflammatory conditions of the musculoskeletal system, such as joint inflammation, and methods of lubricating a joint using the described visco-supplement compositions.

CROSS REFERENCE TO RELATED APPLICATION

This application is entitled to priority pursuant to 35 U.S.C. § 119(e)to U.S. Provisional Patent Application No. 62/083,606, filed Nov. 24,2014, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to visco-supplement compositions derivedfrom a transcellular fluid. The present invention also relates tomethods of treating an inflammatory condition of the humanmusculoskeletal system, such as joint inflammation or osteoarthritis,and increasing joint lubrication with the visco-supplement compositionsdescribed herein.

BACKGROUND OF THE INVENTION

Visco-supplements are viscous substances that can function as alubricant and shock absorber for joints, and are often used to treatinflammatory conditions of the human musculoskeletal system. Manyvisco-supplements typically used for treating inflammatory conditions ofthe human musculoskeletal system are produced via bacterial culturing,or by a process of extraction from tissues of a different species, suchas rooster combs, which is the fleshy growth on top of the rooster'shead.

The primary viscous, protecting, nutritional and lubricating fluid ofthe human musculoskeletal system responsible for maintaining healthy,articulating joints is synovial fluid. Synovial fluid is secreted by thesynovial membrane, which is the soft tissue found between the articularcapsule (joint capsule) and joint cavity. In native joints, synovialfluid functions as a biomechanical lubricant, lowering the friction andwear of articulating cartilage in joints, as well as a source ofnutrients for the articulating tissues. Synovial fluid lubricatingmacromolecules, including hyaluronic acid and proteoglycan 4 (PRG4), aresecreted by synoviocytes in the synovial membrane lining the joint andchondrocytes in the cartilage of the joint, and are concentrated insynovial fluid due to the retaining property of the semi-permeablesynovial membrane.

Currently available visco-supplements for the treatment of inflammatoryconditions of the human musculoskeletal system are comprised ofessentially a single component, hyaluronic acid (HA). Considering thecomplexity of naturally occurring synovial fluid, and the complexbiological system necessary for maintaining the health and properfunctioning of articulating joints, pure HA is not an ideal treatmentfor inflamed joints at least for physiological reasons.

Furthermore, the clinical record of currently available HA basedvisco-supplements is poor. That record was recently reviewed by theAmerican Academy of Orthopedic Surgeons (AAOS), and in June, 2013, theAAOS issued clinical practice guidelines to physicians, whichrecommended against using HA for patients with symptomaticosteoarthritis (OA) of the knee based on supporting evidence fromseveral high-quality research studies that met the inclusion criteria.

The AAOS's clinical practice guidelines are based on some of the bestpeer reviewed study evidence available. According to the AAOS's website,currently published studies do not show a clinically effective responsefor HA injections based on minimal clinically important improvements(MCIIs). Some peer reviewers were critical of the AAOS' findings andrecommendation, especially in light of the important clinical practiceimplications, and highlighted prior systematic reviews supporting theuse of HA. However, these reviews were analyzed and found to haveseveral flaws. For example, most did not address the issues ofpublication bias, between-study heterogeneity, and clinical significancein determining final recommendations.

Inflammation of articulating surfaces of the musculoskeletal system isone of the most common medical complaints. Although the exact causes forpainful knee, hip, shoulder, facet, ankle and wrist joints may bedifficult to ascertain and in many cases are unknown, it is understoodthat degenerative damage, especially cartilage damage, plays a centralrole in the pathogenic mechanism leading to this disorder. Currenttreatment modalities include pharmacological treatments, physiotherapy,visco-supplement injections, corticosteroid injections and, at theterminus of a continuum of care for joint pain, surgical replacement ofthe joint. According to the Centers for Disease Control (CDC), nearlyone in two people are projected to develop symptomatic kneeosteoarthritis by age 85 years; two in three people who are obese areprojected to develop symptomatic knee osteoarthritis in their lifetime;and one in four people are projected to develop hip arthritis in theirlifetime. Also, according to the CDC, an estimated 52.5 million adultsin the United Stated reported being told by a doctor that they have someform of arthritis, rheumatoid arthritis, gout, lupus, or fibromyalgia.

Moreover, by 2030, the number of Americans aged 18 years or older whoare projected to have doctor-diagnosed arthritis is 67 million.

Present pharmacological treatments for such joint inflammation includethe use of non-steroidal anti-inflammatory drugs (NSAIDs), such asnaproxen, ibuprofen, etc., and drugs of the cyclooxygenase-2 inhibitorgroup like celecoxib, as well as other drugs including glucosamine,chondroitin, and opiates. Present non-pharmacological treatments includehot or cold packs around the inflamed joint; anaerobic exercises, suchas resistance training; suggestion of weight loss or use of a crutch;use of a brace, particularly for the patella; and correction of jointtiling or misalignment.

However, many of the pharmacological and non-pharmacological treatmentsemployed for treating joint inflammation suffer from several drawbacks.For example, corticosteroid injections are one of the most commonanti-inflammatory treatments for joint pain, and it carries many risksincluding deteriorating articulating cartilage in the joint if overused,atrophy of subcutaneous fat, and nerve inflammation. There is, as aresult, growing interest in the development of novel technologies torepair or regenerate the painful, degenerated articulatingmusculoskeletal bone and cartilage system.

Accordingly, there exists a need in the art for improved methods andcompositions for treating inflammation of the human musculoskeletalsystem. Preferably, such improved compositions provide the requisitelubrication, cushioning, and protection of the joint, but more closelyresemble the composition of synovial fluid found in healthy articulatingjoints as compared to known visco-supplements.

BRIEF SUMMARY OF THE INVENTION

The present invention satisfies this need by providing avisco-supplement that can be used to treat inflammation of the humanmusculoskeletal system, and particularly joint inflammation. Thevisco-supplement of the present invention, which is a processedtranscellular fluid, provides not only the requisite lubrication,cushioning, and protection of the joint, but also the components and pHlevel required for maintaining the healthy biological and physiologicalfunction of the entire joint system.

In one general aspect, the present invention provides a visco-supplementcomposition. According to embodiments of the present invention, thevisco-supplement composition comprises a processed transcellular fluid,wherein the processed transcellular fluid comprises:

(a) an increased concentration of at least one of a first componentselected from the group consisting of proteins, lipids, andcarbohydrates as compared to a concentration in an unprocessedtranscellular fluid; and

(b) a decreased concentration of at least one of a second componentselected from the group consisting urea, uric acid, and creatinine, ascompared to a concentration in the unprocessed transcellular fluid,

and the composition has a pH in a range of about 6.0 to about 8.0.

In a preferred embodiment, the unprocessed transcellular fluid is ahuman transcellular fluid. In another preferred embodiment, theunprocessed transcellular fluid is selected from the group consisting ofan amniotic fluid (AF), aqueous humor fluid (AHF), and vitreous humorfluid.

In another general aspect, the present invention provides a method oftreating an inflammatory condition of a musculoskeletal system in asubject in need of treatment thereof. According to embodiments of thepresent invention, the method comprises injecting a visco-supplementcomposition according to an embodiment of the present invention into themusculoskeletal system of the subject. Preferably, the inflammation ofthe musculoskeletal system is joint inflammation, and more preferablyinflammation of the knee joint, such as osteoarthritis of the knee.

In a particular embodiment, the present invention provides a method oftreating joint inflammation in a knee in a subject in need thereof, themethod comprising administering to the knee of the subject, avisco-supplment composition comprising a processed human amniotic fluid,wherein the processed human amniotic fluid comprises:

-   -   (a) an increased concentration of at least one of a first        component selected from the group consisting of proteins,        lipids, and carbohydrates as compared to a concentration in an        unprocessed human amniotic fluid; and    -   (b) a decreased concentration of at least one of a second        component selected from the group consisting urea, uric acid,        and creatinine, as compared to a concentration in the        unprocessed human amniotic fluid,        wherein the composition has a pH in a range of about 6.0 to        about 8.0, and wherein the administration comprises        intra-articular injection to the knee.

In another particular embodiment, the subject to be treated hasosteoarthritis, thereby causing the joint inflammation in the knee.

In yet another general aspect, the present invention provides a methodof lubricating a joint in a subject in need thereof. The methodcomprises injecting into an articular capsule of the joint in thesubject a visco-supplement composition according to an embodiment of thepresent. In a preferred embodiment, the joint is a knee joint.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. In this application, certainterms are used, which shall have the meanings as set in thespecification. It must be noted that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein, “visco-supplement” refers to a viscous or gel-like fluidthat can function as a replacement for, or supplement to, a naturalfluid present in a musculoskeletal system, such as a synovial fluid.

As used herein, “transcellular fluid” is the portion of the body waterwhich is contained within epithelial lined spaces. It is anultra-filtrate of blood plasma, and is derived from blood plasma.Examples of transcellular fluids are aqueous humour, blood serum,amniotic fluid, cerebrospinal fluid, sweat, tears, synovial fluid,ocular fluid, pericardial fluid, vitreous humour, bile, saliva, semen,vaginal lubrication, breast milk, mucus, and urine. Transcellular fluidstypically have specialized functions, which depend upon the particularlocation of the body that the transcellular fluid is found.

“Synovial fluid,” as used herein, refers to the viscous fluid found inthe cavities of synovial joints. Synovial fluid is also referred to as“joint fluid.” Synovial fluid is secreted by the synovial membrane,which is the inner membrane lining a synovial joint. The primary role ofsynovial fluid is to reduce friction between articular cartilage ofsynovial joints during movement. Synovial fluid has other functions,including, but not limited to, reducing joint friction throughlubrication, shock absorption, and nutrient and waste transportation.

As used herein, “aqueous humor fluid” generally refers to thetranscellular fluid secreted into the posterior chamber of the eye.“Vitreous humor fluid” generally refers to the fluid found in thevitreous of the eye, which is the space between the lens and retina ofthe eyeball.

As used herein, “amniotic fluid” refers to the fluid contained insidethe membrane that forms a protective sac around the embryo and later thefetus, in a pregnant female. The placenta itself acts as a filter forblood plasma and produces the amniotic fluid.

The term “unprocessed,” when used with respect to a transcellular fluid,refers to a transcellular fluid that has not been treated, such that ithas substantially the same composition, i.e., same components in thesame amounts, as the native transcellular fluid found in vivo.

The term “processed,” when used with respect to a transcellular fluid,means that the transcellular fluid has been treated, such that it has analtered composition as compared to the native transcellular fluid foundin vivo. In one embodiment, a processed transcellular fluid has one ormore different components, such as proteins, lipids, and carbohydrates,as compared to the unprocessed transcellular fluid. In anotherembodiment, a processed transcellular fluid has the same components,such as proteins, lipids, and carbohydrates, as compared to theunprocessed transcellular fluid, but the components are present indifferent amounts, i.e., in an increased concentration or a decreasedconcentration. In another embodiment, a processed transcellular fluidhas a different viscosity as compared to the unprocessed transcellularfluid.

Embodiments of the present invention relate to a visco-supplementcomposition that is an enriched composition of a transcellular fluid foruse in treating an inflammatory condition of the musculoskeletal system,such as joint inflammation, and for lubricating a joint. Thevisco-supplement composition of the present invention more closelyapproximates the composition of synovial fluid as compared toconventional visco-supplements and other injection materials used totreat inflammatory conditions of the musculoskeletal system and jointinflammation.

In one general aspect, the present invention provides a visco-supplementcomposition comprising a processed transcellular fluid. The processedtranscellular fluid can be derived from any transcellular fluiddescribed herein, and is preferably an amniotic fluid, aqueous humorfluid, or vitreous humor fluid.

According to preferred embodiments, the transcellular fluid is a humanamniotic fluid, a human aqueous humor fluid, or a human vitreous humorfluid. In a particularly preferred embodiment, the transcellular fluidis a human amniotic fluid, and more preferably is human amniotic fluidharvested from a pregnant female undergoing either Caesarean delivery orvaginal birth. Preferably, the technique used for harvesting theamniotic fluid should substantially eliminate, or at least minimize, thepresence of red blood cells in the amniotic fluid. Furthermore, theamniotic fluid used in the present invention should not be cloudy incolor, and it should not have any particulate matter.

According to embodiments of the present invention, the concentrations ofthe components found in the unprocessed transcellular fluid,particularly inorganic constituents, gases, non-protein nitrogenouscompounds, proteins, carbohydrates, and lipids, differ from theconcentrations found in the processed transcellular fluid. Theconcentration of any one particular component in the processedtranscellular fluid can be increased, decreased, or unchanged ascompared to the concentration in the unprocessed transcellular fluid,independent of any other component. For example, the concentration ofcreatinine in the processed transcellular fluid can be decreased,whereas the concentration of glucose can be increased, as compared totheir respective concentrations in the unprocessed transcellular fluid.

According to embodiments of the present invention, the processedtranscellular fluid comprises an increased concentration of at least oneof a first component selected from the group consisting of proteins,lipids, and carbohydrates as compared to a concentration in anunprocessed transcellular fluid; and a decreased concentration of atleast one of a second component selected from the group consisting urea,uric acid, and creatinine, as compared to a concentration in theunprocessed transcellular fluid.

In one general aspect a visco-supplement composition according to thepresent invention comprises a processed transcellular fluid havingincreased concentrations of proteins as compared to the unprocessedtranscellular fluid. According to embodiments of the present invention,the total protein concentration is increased in the processedtranscellular fluid, such that the aggregate protein content ranges fromabout 30.25 g/L to about 50.375 g/L. The concentrations of at least oneof cytokines, such as interleukin (IL)-6, IL-8, IL-10, and tumornecrosis factor (TNF)-α, are increased in the processed transcellularfluid; and the concentrations of at least one of globulins, such asα-globulins, β-globulins, and γ-globulins, are increased as compared totheir concentrations in the unprocessed transcellular fluid. Accordingto other embodiments of the invention, albumin comprises no less than60% of the total protein content in the processed transcellular fluid.Also according to embodiments of the present invention, theconcentration of other proteins present in the unprocessed transcellularfluid, such as C-reactive protein, procalcitonin, and/or calprotecin canbe present in the processed transcellular fluid, or they can beeliminated.

In preferred embodiments of the present invention, in the processedtranscellular fluid, the concentration of α-globulins is at least 12% ofthe total protein concentration, preferably 12% to 18%; theconcentration of β-globulins is at least 16% of the total proteinconcentration, preferably 16% to 27%; and the concentration ofγ-globulins is at least 12% of the total protein concentration,preferably 12% to 18%. In other preferred embodiments, in the processedtranscellular fluid, the concentration of IL-6 is at least 329 ng/L, theconcentration of IL-8 is at least 421 ng/L, the concentration of IL-1βisat least 3.9 ng/L and the concentration of TNF-α is at least 11.5 ng/L.

In another general aspect, a visco-supplement composition according tothe present invention comprises a processed transcellular fluid havingincreased concentrations of lipids as compared to the unprocessedtranscellular fluid. According to embodiments of the present invention,the concentrations of at least one of fatty acids, cholesterol, andphospholipids in the processed transcellular fluid are increased ascompared to their respective concentrations in the unprocessedtranscellular fluid. In preferred embodiments of the invention, theprocessed transcellular fluid comprises fatty acids at a concentrationof about 0.25 g/L to about 6.5 g/L; cholesterol at a concentration ofabout 0.5 g/L to about 9.5 g/L; and/or phospholipids at a concentrationof about 0.003 g/L to about 0.3 g/L.

In yet another general aspect, a visco-supplement composition accordingto the invention comprises a processed transcellular fluid havingincreased concentrations of carbohydrates as compared to the unprocessedtranscellular fluid. According to embodiments of the present invention,the concentrations of one or more of glucose, fructose, hyaluronic acid,and lubricin in the processed transcellular fluid are increased ascompared to their respective concentrations in the unprocessedtranscellular fluid. Lubricin (proteoglycan 4) is a proteoglycan presentin synovial fluid that acts as lubricant. These carbohydrates functionto provide lubrication, protection and shock absorption, particularly toinflamed joints. In preferred embodiments of the present invention, theprocessed transcellular fluid comprises glucose at a concentration ofabout 600 mg/L to about 900 mg/L; fructose at a concentration of about48 mg/L to about 59 mg/L; hyaluronic acid at a concentration of about 30μg/L to about 3600 μg/L; and/or lubricin at a concentration of about 10μg/ml to about 200 μg/ml.

And in yet another general aspect, a visco-supplement compositionaccording to the present invention comprises a processed transcellularfluid having decreased concentrations of non-protein nitrogenouscompounds as compared to the unprocessed transcellular fluid. Inparticular, the concentrations of one or more of urea, uric acid, andcreatinine in the processed transcellular fluid are decreased ascompared to their respective concentrations in the unprocessedtranscellular fluid. According to preferred embodiments of the presentinvention, the concentration of urea is no more than 160 mg/L, theconcentration of uric acid is no more than 80 g/L, and/or theconcentration of creatinine is no more than 14 mg/L in the processedtranscellular fluid used in the visco-supplement of the presentinvention.

According to embodiments of the present invention, the pH of avisco-supplement composition ranges from a pH of about 6.0 to a pH ofabout 8.0, such as 6.0, 6.5, 7.0, 7.5, or 8.0. Preferably, the pH rangesfrom 6.5 to 7.5, and is more preferably 7.0.

Embodiments of the present invention also relate to visco-supplementcompositions comprising a processed transcellular fluid having optimizedconcentrations of inorganic constituents and optimized partial pressuresof gases, such that the concentrations and partial pressures closelyapproximate the concentrations of these components in the nativesynovial fluid. Such inorganic constituents include sodium, potassium,and chloride, and such gases include oxygen and carbon dioxide.

The visco-supplement compositions of the present invention can furthercomprise additional substances including pharmaceutically acceptableexcipients, such as thickeners, salts, preservatives, colorants, etc.;substances to prevent the growth of microbes, such as antifungal,antibacterial, or antiviral agents; and agents that improve theviscosity or thickness of the composition. These additions can be made,provided that they do not cause irritation of the joint, or interferewith the healing properties of the transcellular fluid. Thevisco-supplement compositions of the present invention can also furthercomprise one or more pharmaceutically active ingredients, such as ananalgesic, an anti-inflammatory agent, an anti-microbial agent, etc.

According to embodiments of the present invention, the visco-supplementcompostion can further comprise a cryoprotectant. Any cryoprotectantsuitable for pharmaceutical use known to those skilled in the art inview of the present disclosure can be used in the composition of thepresent invention. Examples of cryoprotectants that can be used in thevisco-supplement compositions of the present invention include, but arenot limited to, dimethyl sulfoxide (DMSO), sucrose, glycerol, glucose,and any other sugars, e.g., monosaccharides or disaccharides, alcoholsand penetrating agents, or combinations thereof, routinely used ascryoprotectants by those skilled in the art, which will be know to thoseskilled in the art in view of the present disclsoure. In certainembodiments of the invention, the visco-supplement composition iscryopreserved.

A visco-supplement composition according to a preferred embodiment ofthe present invention is shown in Table 1 below. The transcellular fluidused in the composition is processed human amniotic fluid.

TABLE 1 Concentrations of inorganic constituents, gases, proteins,carbohydrates, lipids, and non- protein nitrogenous compounds in avisco-supplement composition according to an embodiment of the presentinvention. Unprocessed Composition Target Transcellular Fluid¹ Rangespost-processing Component (Amniotic Fluid) High Low Discussion andFunction Inorganic Constituents Sodium 127 mEq/L 148.375 85.625 Staywithin proscribed range Potassium 4.0 mEq/L 48.125 21.875 Increase fromabout 4.0 mEq/L in source material to a minimum of 21.875 mEq/L Chloride105 mEq/L 145.75 66.25 Stay within proscribed range Calcium 4.0 mEq/L7.21875 3.28125 Stay within proscribed range Magnesium 1.4 mEq/L 2.751.25 Stay within proscribed range Phosphorus 29.0 mg/L 55 25 Increasethe amount of phosphorus to the proscribed range Gases/H+ pH 7.0 7.93756.125 In order to restore the proper acid/base balance in an acidic andarthritic joint, stay within the proscribed range PO2 2-15 mm Hg 78 8 inorder to provide the proper oxygenation range for use as a visco- PCO257 mm Hg 150 34 supplement in an arthritic joint, increase to the twoproscribed ranges Protein (Total) 22-31 g/L 50.375 30.25 Increase theamount of protein to the proscribed range Albumin 60% 60% Ensure thatAlbumin comprises no less than 60% of the protein content α-globulins12% 18% 12% Stay within proscribed range β-globulins 16% 27% 16% Staywithin proscribed range γ-globulins 12% 18% 12% Stay within proscribedrange Cytokines IL-6 329 ng/L 329 ng/L No less than the proscribedamount IL-8 421 ng/L 421 ng/L No less than the proscribed amount IL-1β3.9 ng/L 3.9 ng/L No less than the proscribed amount TNF-α 11.5 ng/L11.5 ng/L No less than the proscribed amount C-reactive protein 5.4 mg/LMay be present or may be eliminated (CRP) in any embodiment of theinvention Procalcitonin 1.8 μg/L May be present or may be eliminated inany embodiment of the invention Calprotecin 3425 μg/L May be present ormay be eliminated in any embodiment of the invention Non-ProteinNitrogen These by-products of metabolism Compounds are reduced in thefinal supplement fluid. Urea 370 mg/L 160 82.5 Reduce quantities suchthat the amounts are no more than the maximum amount proscribed UricAcid 50 mg/L 80 22.5 Reduce quantities such that the amounts are no morethan the maximum amount proscribed Creatinine 28 mg/L 14 8.75 Reducequantities such that the amounts are no more than the maximum amountproscribed Carbohydrates These carbohydrates are important to supplyproper levels of nutrients, lubrication, compression and shearresistance and cushioning to the living musculoskeletal joint Glucose330 mg/L 900 600 Increase quantity to the proscribed ranges Fructose 35mg/L 58.625 48.125 Increase quantity to the proscribed ranges LacticAcid 370-750 mg/L 250 156.25 Reduce quantities such that the amounts areno more than the maximum amount proscribed Pyruvate 8 mg/L 13.4 11Increase quantity to the proscribed ranges Hyaluronan: mean22.67(10.8)/1.1(0.46) 3600 μg/ml 30 μg/L Provides Compression & Shear(SD) μg/L Resistance and is a carrier for surface active phospholipids(SAPL), which are a boundary lubricant in visco- supplement fluids.Increase to levels within the proscribed levels. Lubricin (PRG4) 200μg/ml 10 μg/ml This is a protein which is related to MCF and is likelycarrier for SAPL. Increase to the proscribed levels. Total Lipids 0.48g/L Fatty Acids 0.24 g/L 6.50 g/L 0.25 g/L Increase quantity to theproscribed ranges Cholesterol 0.02 g/L 9.50 g/L 0.50 g/L Increasequantity to the proscribed ranges Phospholipids (Total) 0.03 g/L 0.30mg/ml 0.003 g/L Increase quantity to the proscribed ranges ¹References(1)-(10) were used to determine the quantities of the components in theunprocessed amniotic fluid

The transcellular fluid used to produce the visco-supplementcompositions of the present invention is isolated or obtained from adonor. As used herein, the term “donor” refers to a mammal from which atranscellular fluid has been obtained or will be obtained. The term“mammal” as used herein, encompasses any mammal. Examples of mammalsinclude, but are not limited to, cows, horses, sheep, pigs, cats, dogs,mice, rats, rabbits, guinea pigs, monkeys, humans etc., more preferablya human.

According to preferred embodiments of the present invention, a donor isa human, bovine, or porcine, most preferably a human.

Methods of obtaining a transcellular fluid, such as a human amnioticfluid, or a human aqueous humor or vitreous humor fluid, from a donorusing the appropriate sterile techniques are well known to those ofordinary skill in the art. One of ordinary skill in the art would alsobe familiar with procedures for safely and humanely obtaining thetranscellular fluid. For example, human amniotic fluid can be obtainedfrom a donor who is undergoing an amniocentesis procedure, Caesareansection delivery, or vaginal birth using a specially designed receptacleto collect the fluid after rupture of membranes. Amniotic fluid isolatedfrom a donor undergoing a Caesarean delivery is preferred, because redblood cell count can be minimized in this way. However, amniotic fluidobtained from a donor undergoing vaginal birth or Caesarean section canbe used with the present invention. Other techniques for isolatingtranscellular fluids, such as ocular fluid or cerebrospinal fluidinclude simple needle aspiration.

According to embodiments of the present invention, the transcellularfluid is free of particulate matter, such as cellular debris and tissuedebris. Particulate matter can be removed from the transcellular fluidby any method known in the art for removing particulate matter frombiological samples, including but not limited to filtration andcentrifugation. Particulate matter can be removed at any time after thetranscellular fluid has been collected. Preferably, the particulatematerial is removed prior to any other processing or treatment steps.

According to embodiments of the present invention, the processedtranscellular fluid is prepared from an unprocessed transcellular fluid,e.g., by removing non-protein nitrogenous compounds, such as uric acid,urea, creatinine, and nitrogen. These non-protein nitrogenous compoundscan be removed by any method known in the art, such as by means of asemi-permeable membrane or filter, or by dialysis. Other suitable meansfor removing these compounds include osmotic, centrifugal, gravitationalor mechanical pumping forces. The transcellular fluid can also beprocessed to increase the concentration of one or more of proteins,carbohydrates, lipids, and other desirable components using anytechnique known to those of ordinary skill in the art in view of thepresent disclosure. For example, a desirable endogenous component can beenriched by filtration or centrifugation with specific parameters. Anexogenous desirable component can also be added to the processedtranscellular fluid.

For example, using an ultrafiltration approach, a semi-permeablecontainer is filled with raw transcellular fluid, and then a pressuregradient is applied across the semi-permeable membrane using any numberof techniques known to those skilled in the art including, but notlimited to, a high permeability dialyzer. As another illustrativeexample, when employing hemodialysis techniques, an electrolyte solution(dialysate) can be applied on one side of a membrane, creating aconcentration gradient, which causes water and other non-proteincellular components of the transcellular fluid to flow through thesemi-permeable membrane. As yet another illustrative example, rapidultrafiltration approaches can be used. Rapid ultrafiltration approachesemploy a semi-permeable membrane cylindrical container that rotatesconstantly in order to avoid filter clogging even as a pressure gradientis applied to the contained fluid- either from within the container(pushing), or from the opposite side of the semi-permeable membrane(pulling).

The transcellular fluid can also be concentrated by removal of waterusing any technique known to those of ordinary skill in the art. Forexample, substantially all of the water can be removed bylyophilization, etc., or the amount of water can simply be reduced byvacuum filtration, etc.

The transcellular fluid used in the present invention can be furthertreated in order to promote preservation, lengthen shelf life, etc.These treatments include, but are not limited to sterilization, e.g.gamma-irradiation; and cooling, refrigeration and freezing, addition ofone or more preservatives, antimicrobial agents, etc.

Risk assessment is conducted on potential donors with informed consentprior to collection of a transcellular fluid to evaluate the donor'srisk factors for communicable diseases such as such as humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus(HCV), cytomegalovirus (CMV), human T-lymphotropic virus (HTLV),syphilis, etc. Potential donors with high risk factors for thecommunicable diseases are excluded. For example, if an amniotic fluid isto be obtained, risk assessment can be conducted on a pregnant female toevaluate her risk factor for communicable diseases. Medical and socialhistories of the pregnant female, including physical exam record, and/orrisk assessment questionnaire are reviewed, and pregnant females withhigh risk factors for the communicable diseases are excluded.

Consent to draw blood at the time of obtaining the transcellular fluid,and 1 to 12 months after the transcellular fluid is initially collectedfrom the donors with low risk factors for communicable diseases isobtained. Screening tests on communicable diseases, such as HIV 1 and 2,HCV, Hb Core, syphilis, HTLV I/II, CMV, hepatitis B and C, can beconducted by conventional serological tests on the blood sample drawn atthe time the transcellular fluid is obtained. These initial screeningtests are preferably completed within seven days after obtaining thetranscellular fluid. Preferably, the screening tests are conducted againon a second blood sample collected a few months after the transcellularfluid is obtained, to verify the previous screening results and to allowfor detection of communicable diseases acquired shortly before obtainingthe transcellular fluid, but tested as “negative” on the previousscreening tests. The second blood sample can be collected one to twelvemonths after obtaining the transcellular fluid, and is preferablycollected six months after obtaining the transcellular fluid.

According to preferred embodiments, only donors with informed consentwho tested negative for communicable diseases are approved astranscellular fluid donors. According to other preferred embodiments,only donors with informed consent who tested negative for thecommunicable diseases in both screening tests with blood sample drawn atthe time the transcellular fluid is obtained and blood sample drawn oneto twelve months after the transcellular fluid is obtained, preferablysix months after, are approved as transcellular fluid donors.

The unprocessed transcellular fluid for use in the present invention canalso be screened after collection to ensure that it is not contaminatedwith any communicable disease agents, such as human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV),cytomegalovirus (CMV), human T-lymphotropic virus (HTLV), syphilis, etc.

Embodiments of the present invention also relate to methods of treatinginflammatory conditions of the musculoskeletal system, and methods ofincreasing joint lubrication using a visco-supplement compositionaccording to an embodiment of the present invention.

According to embodiments of the present invention, a method of treatingan inflammatory condition of a musculoskeletal system comprisesinjecting a visco-supplement composition as described herein into themusculoskeletal system of a subject, such as injection into a joint.Examples of joints into which a visco-supplement composition of thepresent invention can be injected include all synovial joints, such ashinge joints (e.g., elbow and knee), pivot joints (e.g., atlas and axisbones at the top of the neck), ball and socket joints (e.g., hip),saddle joints (e.g., carpometacarpal joint of the thumb), condyloidjoints (e.g., wrist, metacarpophalangeal joints, metatarsophalangealjoint), and gliding joints (e.g., intercarpal joints in the wrist).Other examples of joints into which a visco-supplement composition ofthe present invention can be injected include all amphiarthoses joints,such as intervertebral discs.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human, to who will be or has been administered avisco-supplement composition according to the present invention.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to an inflammatory condition ofthe musculoskeletal system, which is not necessarily discernible in thesubject. The terms “treat,” “treating,” and “treatment” can also referto preventing the progression, or at least slowing down the progressionof the inflammatory condition of the musculoskeletal system. In oneembodiment, “treat,” “treating,” and “treatment” refer to a reduction orcomplete alleviation of pain associated with the inflammatory conditionof the musculoskeletal system. In another embodiment, “treat,”“treating,” and “treatment” refer to a reduction of joint inflammation.In yet another embodiment, “treat,” “treating,” and “treatment” refer toan alleviation of one more symptoms associated with joint inflammation,such as joint pain, joint swelling, joint stiffness, and difficulty injoint movement.

As used herein “an inflammatory disorder of the musculoskeletal system”refers to any disease, disorder, or condition characterized byinflammation of a part of the musculoskeletal system. Themusculoskeletal system is comprised of joints, tendons, bone, andcartilage. Examples of inflammatory diseases of the musculoskeletalsystem include, but are not limited to joint inflammation, such asarthritis, particularly osteoarthritis and rheumatoid arthritis;bursitis; bone infection (osteomyelitis); tendonitis; inflammation ofthe cartilage, such as chondromalacia patellae; and chronic conditionsthat cause inflammation of the musculoskeletal system, and particularlyjoints, such as fibromyalgia and Lupus.

According to preferred embodiments of the present invention, theinflammatory disorder of the musculoskeletal system to be treated isjoint inflammation. In a particularly preferred embodiment, the jointinflammation is inflammation of a synovial joint, such as a knee joint,a wrist joint, a shoulder joint, a hip joint, or an elbow joint, and ismost preferably a knee joint. Because the visco-supplement compositionsof the present invention contain many of the same components as thesynovial fluid found in healthy joints, the compositions areparticularly suited for covering, lubricating, protecting and relievingjoint inflammation when injected into the intra-articular space of thejoint.

A method of lubricating a joint according to embodiments of the presentinvention comprises injecting a visco-supplement composition asdescribed herein into an intra-articular space of the joint. Theintra-articular space refers to the space inside of a joint between twobones that is usually contained by the articular capsule. The articularcapsule, also referred to as the joint capsule, is the envelopesurrounding a synovial joint. Preferably the joint to be lubricated is asynovial joint, and is more preferably a knee joint. In a preferredembodiment, a subject is in need of joint lubrication to reduce painassociated with joint movement. In another embodiment of the presentinvention, the visco-supplement composition is applied to the subject incombination with another treatment for the inflammatory disorder of themusculoskeletal system. Examples of other treatment for the inflammatorydisorder of the musculoskeletal system that can be used in combinationwith the visco-supplement composition include, but are not limited to,general exercise; strengthening exercises; walking aids such as canes orcrutches; wheeled walkers or frames; knee bracing; orthotics;non-steroidal anti-inflammatory drugs (NSAIDs); thermal treatmentsincluding heat therapy; cryotherapy; transcutaneous electrical nervestimulation; and acupuncture.

In a particular embodiment, the present invention provides a method oftreating joint inflammation in a knee in a subject in need thereofcomprising administering a visco-supplement composition of the presentinvention, wherein the administration comprises intra-articularinjection to the knee. Any of the visco-supplement compositionsdescribed herein can be used, and preferably the visco-supplementcomposition comprises a processed human amniotic fluid.

According to embodiments of the invention, the visco-supplementcomposition can be cryopreserved prior to administration, e.g.,injection. Any of the methods of treating an inflammatory condition of amusculoskeletal system, e.g., joint inflammation and particularly jointinflammation of the knee, and any of the methods of lubricating a jointin a subject according to embodiments of the present invention, whereinthe visco-supplement composition is cryopreserved prior to injection,can further comprise a step of warming the visco-supplement compositionto a temperature in a range of about 15° C. to 25° C., such as 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25° C. before administering thecomposition to the subject. Preferably, the composition is warmed to atemperate of about 20° to 22° C.

According to embodiments of the present invention, the visco-supplementcompositions described herein can be used directly in a method of thepresent invention. Alternatively, the visco-supplement compositions canbe diluted by about 10%-50%. Any diluent known to those of ordinaryskill in the art can be used to dilute the visco-supplement composition,such as physiologically compatible saline solution, balanced salinesolution, sodium hyaluronate, methylcellulose, dimethylsulfoxide, or anyother cryopreservant.

The following examples of the present invention are to furtherillustrate the nature of the invention. It should be understood that thefollowing examples do not limit the invention and that the scope of theinvention is to be determined by the appended claims.

EXAMPLES Example 1 Clinical Study Evaluating the Efficacy of aVisco-Supplement Composition of the Present Invention in TreatingOsteoarthritis

Approximately two hundred seventy five (275) patients diagnosed withosteoarthritis of the knee each received a single injection of avisco-supplement composition derived from a human amniotic fluidaccording to the present invention. The visco-supplement composition wascryopreserved at −150° C., and then warmed to about 20° C. to 22° C. inthe clinic prior to injection. The visco-supplement composition (4 mL)was then injected into the articular capsule of the knee joint. The painlevel of each of the patients was determined at intervals of thirtydays, ninety days, and six months after the initial injection, asmeasured by the Visual Analog Scale (VAS) and Western Ontario andMcMaster Universities Arthritis Index (WOMAC). VAS and WOMAC are a setof standardized questions widely used by health professionals toevaluate the condition of patients with osteoarthritis of the hip andknee, by asking a set of questions to assess pain, stiffness, andphysical function.

The results of the clinical study are shown below in Table 2, referredto as “Study A.” Also shown in Table 2 are the reported results ofsimilar studies conducted with non-human derived hyaluronic acid(Studies B, C, and D), and corticosteroid visco-supplements (Studies E,F, and G) for comparison. In the reported studies, pain relief from thehyaluronic acid visco-supplement and corticosteroid injections was alsoevaluated using VAS and WOMAC. Briefly, the reported data in the Tablefor Studies B-G was obtained as follows: Study B was the therapeutictrajectory following intra-articular hyaluronic acid injection in kneeosteoarthritis (meta-analysis); Study C evaluated the efficacy of asingle intra-articular injection of Gel-200 (cross-linked formulation ofhyaluronic acid) in treating symptomatic knee osteoarthritis in amulticenter randomized controlled trial; Study D evaluated the efficacyof Sinovial® (purified intra-articular hyaluronic acid) and Synvisc®(hylan G-F20) in the treatment of symptomatic knee osteoarthritis; StudyE evaluated the efficacy of intra-articular corticosteroid injections intreating knee osteoarthritis; Study F evaluated pain relief aftersteroid injection in the knee of patients with rheumatoid knee; andStudy G was a randomized trial evaluating the efficacy ofintra-articular injections of a composition of hyaluronic acid,corticosteroid, and isotonic saline in treating hip osteoarthritis.

TABLE 2 Comparison of pain relief and adverse events experienced bypatients having joint inflammation and treated with injections of eithera visco-supplement composition of the present invention, a hyaluronicacid derived injection, or corticosteroid injection. Processed AmnioticFluid Hyaluronic Acid Visco-supplement (non-human derived)Corticosteroid Injection of the invention Injection Studies StudiesStudy A Study B Study C Study D Study E Study F Study G Number of 275 44379 381 28 RCT 300 101 patients (1973 patients)¹ % pain 61%% 31% 41% 54%22% 18% 27% relief (at 30 days) (at 30 days) (overall) (overall)(overall) (overall) (overall) (VAS, 53% WOMAC) (at 180 days) AdverseNone reported N/A Joint Injection site None None None Events swelling,hematoma, reported reported reported effusions, injection site,arthralgia pain, arthralgia, and joint swelling ¹”28 RCT (1973patients)”-refers to twenty-eight randomized control tests (RCT)representing an aggregate of 1,973 patients.

From the results of the clinical study reported in Table 2, it can beseen that the visco-supplement composition according to an embodiment ofthe present invention was highly efficacious in relieving pain inpatients with knee osteoarthritis, with patients reporting 61%improvement in pain thirty days after the initial injection. The painrelief continued with an average 53% rate of pain relief after sixmonths following the initial injection. Importantly, 84.65% of thepatients reported 40% or greater pain improvement based on total WOMACscore at 30 days. The visco-supplement composition of the presentinvention was also highly tolerated by patients, as evidenced by thefact that adverse events were 2.5%, but were rleated to injection sitepain and resolved rapidly. Moreover, comparing the results of the studywith the visco-supplement composition of the present invention to thoseof studies conducted with non-human derived hyaluronic acid andcorticosteroid injections, the composition of the present invention hasincreased efficacy and tolerability in patients.

The results of the clinical study indicate that the visco-supplementcomposition of the present invention is highly efficacious for treatinginflammation of the musculoskeletal system, and particularly jointinflammation. The results also indicate that the visco-supplementcomposition is highly tolerated in patients, with low adverse sideeffects reported up to thirty days after the initial injection.

Accordingly, the visco-supplement composition of the present inventionprovides a promising new treatment for joint inflammation havingimproved safety and efficacy.

Example 2 Additional Clinical Studies Evaluating the Efficacy of aVisco-Supplement Composition of the Present Invention in TreatingOsteoarthritis

This study included one hundred sixty two (162) patients diagnosed withGrade 1-3 knee osteoarthritis as determined using the radiologicKellgren-Lawrence scale. The Kellgren-Lawrence scale is a scoring toolused to assess the severity of knee osteoarthritis on a radiograph. Ascore of Grade 0 indicates no radiographic features of osteoarthritis;Grade 1 indicates possible joint space narrowing and osteophyteformation; Grade 2 indicates definite osteophyte formation with possiblejoint space narrowing; Grade 3 indicates multiple osteophytes, definitejoint space narrowing, sclerosis, and possible bony deformity; and Grade4 indicates large osteophytes, marked joint space narrowing, severesclerosis and definite bony deformity.

In this study, each patient received a single injection of avisco-supplement composition derived from a human amniotic fluidaccording to the present invention. The visco-supplement composition wascryopreserved at −150° C., and then warmed about 20° C. to 22° C. in theclinic prior to injection. The visco-supplement composition (4 mL) wasthen injected into the articular capsule of the knee joint. The painlevel of each of the patients was determined at intervals of thirtydays, ninety days and six months after the initial injection, asmeasured by the Visual Analog Scale (VAS) and Western Ontario andMcMaster Universities Arthritis Index (WOMAC).

VAS and WOMAC was measured at baseline, and again at ninety days (90days) after the initial injection. The results of this study arereported as percent average improvement. The WOMAC results, which areshown in Table 3 below, are reported separately for two subscales (WOMACPain 500 mm, and WOMAC Difficulty 1700 mm), and also for the sum ofthese two subscales (WOMAC 2400 mm). The absolute average values forbase-line and improvement after 90 days for the VAS and WOMAC scales arereported in millimeters (mm), which is customary practice forresearchers and clinicians.

TABLE 3 WOMAC and VAS scores reported 90 days after initial injection(values are the average of data from 162 patients total) 90- day (t₉₀)Results After Initial Injection 90 day (t₉₀) 90 day (t₉₀) 90 day (t₉₀)90 day (t₉₀) Avg % % over 40% % over 75% 90 day (t₉₀) Avg improvedimproved improved improved Avg from t₀ from t₀ from t₀ from t₀ WOMAC366.17 ± 473.43 −686.91 ± 501.75 65.23% 82.10 54.94 (2400 mm) 153(0-2246) −590.5 (−2122-445) WOMAC 256.05 ± 336.30 −486.29 ± 367.6265.51% 81.48 55.56 DIFFICULTY 99.5 (0-1616) −435.5 (1533-305) (1700 mm)WOMAC 34.64 ± 46.48 −64.02 ± 54.05 64.90% 79.63 61.11 STIFFNESS 14(0-197) −67 (−181-106) (200 mm) WOMAC 75.48 ± 98.76 −136.60 ± 111.0064.41% 77.78 54.94 PAIN 34.5 (0-433) −109 (−459-119) (200 mm) VAS 20.33± 24.16 −34.89 ± 27.39 63.18% 75.93 54.32 10.5 (0-100) −36 (100-36)

As shown by the data in Table 3 above, ninety days after treatment asignificant number of patients participating in the study reported over40% improvement (over 80% as determined by aggregated WOMAC scores; and76% as determined by VAS scores).

Moreover, only 4 of the 162 total patients reported any adverse sideeffects after 90 days. Arhraligia (pain in the knee joint) is the mostcommon treatment-related side effect in hyaluronic acid studies, and istypically reported as an acute (immediate) or near-acute adverse event.In contrast to the observations in the clinical study with thevisco-supplement composition of the present invention, such adverseevents have typically been reported in treatment studies with othervisco-supplement compositions, such as, or example Orthovisc (12.6%),Euflexxa (9.2%), GelOne (17.7%), Monovisc (5.0%), and Synvisc (7.0%),Durolane/Supartz (17.2%) (data obtained from online PubMed Database ofClinical Studies).

Taken together, the above results demonstrate that compositions of thepresent invention can be used to effectively treat diseases, disorders,and conditions of the musculoskeletal system, such as osteoarthritis,with very low reported adverse side effects, and with significantimprovement observed only a few weeks after a single injection.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

REFERENCES

-   -   1. Assali N A: Biology of Gestation, Vol 1, p 276, New York,        Academic Press, 1968 unless noted otherwise    -   2. Engstrom-Laurent A and T C Laurent. Clinical Impact of Bone        and Connective Tissue Markers Pg 237 Academic Press 1989 (AF        values are for 16 weeks and 39 weeks gestation, respectively)    -   3. Laurent U B G, R K Reed. Advance Drug Delivery Reviews        7:237-256 (1991)    -   4. Cajori A F. J Biol Chem. 76:471-480 (1928)    -   5. Treuhaft P S and D J McCarty Arthritis & Rheumatism        14(4):475-484 (1971)    -   6. Osteoarthritis: Diagnosis and Medical/Surgical Management        (Moskowitz ed.), pg 207 Lippincott Williams & Wilkins (2007)    -   7. Bole G G. Arthritis and Rheumatism 5(6):589-601 (1962)    -   8. Wilson S E et al. IOVS 30(3):449-453 (1989)    -   9. Cenedella R J. Biochimica et Biophysic Acta 793:448-454        (1984)    -   10. Schmidt T A et al. JAMA Ophthalmol 131(6):766-776 (2013)    -   11. Murphy et al. Arthritis Rheum 2008; 59(9):1207-1213.    -   12. Murphy et al. Osteoarthritis Cartilage 2010; 18(11):1372-9.

I claim:
 1. A visco-supplement composition comprising a processedtranscellular fluid, wherein the processed transcellular fluidcomprises: (a) an increased concentration of at least one of a firstcomponent selected from the group consisting of proteins, lipids, andcarbohydrates as compared to a concentration of the first component inan unprocessed transcellular fluid; and (b) a decreased concentration ofat least one of a second component selected from the group consistingurea, uric acid, and creatinine, as compared to a concentration of thesecond component in the unprocessed transcellular fluid, and thecomposition has a pH in a range of about 6.0 to about 8.0, wherein theprocessed transcellular fluid includes 30.25 g/L to 50.375 g/L ofproteins, 0.25 g/L to 6.5 g/L of fatty acids, 0.5 g/L to 9.5 g/L ofcholesterol, 0.003 g/L to 0.3 g/L of phospholipids, glucose at aconcentration of 600 mg/L to 900 mg/L, fructose at a concentration of 48mg/L to 59 mg/L, hyaluronic acid at a concentration of 30 μg/L to 3600μg/L,, lubricin at a concentration of 10 μg/ml to 200 μg/ml, no morethan 160 mg/L of urea, no more than 80 g/L of uric acid and no more than14 mg/L of creatinine.
 2. The visco-supplement composition according toclaim 1, wherein the unprocessed transcellular fluid is a humantranscellular fluid.
 3. The visco-supplement composition according toclaim 1, wherein the unprocessed transcellular fluid is selected fromthe group consisting of an amniotic fluid, aqueous humor fluid, andvitreous humor fluid.
 4. The visco-supplement composition according toclaim 1, further comprising a cryoprotectant.
 5. The visco-supplementcomposition according to claim 4, wherein the cryoprotectant is selectedfrom the group consisting of dimethylsulfoxide (DMSO), glucose, sucrose,glycerol, and combinations thereof.
 6. The visco-supplement compositionaccording to claim 1, wherein the first component is a protein selectedfrom the group consisting of interleukin (IL)-6, IL-8, IL-10, and tumornecrosis factor (TNF)-α.
 7. The visco-supplement composition accordingto claim 1, wherein the first component is a lipid selected from thegroup consisting of fatty acids, cholesterol, and phospholipids.
 8. Thevisco-supplement composition according to claim 1, wherein the firstcomponent is a carbohydrate selected from the group consisting ofglucose, fructose, hyaluronic acid, and lubricin.
 9. Thevisco-supplement composition according to claim 1, wherein thecomposition is substantially free of particulate matter.
 10. Thevisco-supplement of claim 1 wherein the processed transcellular fluidincludes 48.125 mg/L to 58.625 mg/L of fructose.
 11. Thevisco-supplement of claim 1 wherein the processed transcellular fluidincludes 156.25 mg/L to 250 mg/L of lactic acid.
 12. Thevisco-supplement of claim 1 wherein the processed transcellular fluidincludes 11 mg/L to 13.4 mg/L of pyruvate.
 13. The visco-supplement ofclaim 1, wherein the processed transcellular fluid includes at least 329ng/L of IL-6, at least 421 ng/L of IL-8, at least 3.9 ng/L of IL-10 andat least 11.5 ng/L of TNF-α.